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Enhancement of mitogen-induced lymphocyte proliferation after preincubation is due to altered sensitivity to prostaglandins
Immunology Letters, 4 (1982) 179-183
Elsevier BiomedicalPress
ENHANCEMENT OF MITOGEN-INDUCED LYMPHOCYTE PROLIFERATION AFTER P R E I N C U B A T I O N IS DUE T O A L T E R E D S E N S I T I V I T Y T O P R O S T A G L A N D I N S L. KALMAR and P. GERGELY 2nd Department o f Medicine, Semmelweis University, Budapest, Hungary
(Received 1 September 1981) (Accepted 5 December 1981)
1. Summary Twenty-four hour preincubation at 37°C enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolishes the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37°C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37°C, PGE~ elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGEI and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37°C.
There is a large body of evidence on the immunological effects of prostaglandins. The prostaglandins may have a direct role in the regulation of immunological processes [9,21,22]. A connection has been assumed between the prostaglandin-producing suppressor cell function and the so-called "short-lived suppressor cell" phenomenon [10]. Prerequisites of the regulatory effects of prostaglandins are the following: (I) immunocompetent cells are able to produce prostaglandins (mostly PGE1 and PGE2), and the rate of production can be influenced by antigens and mitogens [6,16,19,25 ]; and (2) the effector cell functions can be altered by the produced prostaglandins because these cells have specific prostaglandin receptors in their plasma membranes. The PGE~ receptor function of human peripheral blood mononuclear leukocytes was investigated after 24 h preincubation at 37°C. The preincubation technique was identical to that used in the "short-lived suppressor cell" test [2,5]. Our aim was to study the effect of preincubation on the "spontaneous" intracellular cyclic adenosine-3',5'-monophosphate (cAMP) level and cAMP level of cells treated with PGE1 and the connection of the above parameters with the change of mitogen-induced lymphocyte proliferation after preincubation.
3. Materials and methods
2. Introduction kymphocytes have receptors for many hormones and hormone4ike substances [7,17,18,26,27,29]. Their function and biological role are not well known. 0165-2478/82/0000-0000/$2.75 © ElsevierBiomedicalPress
3.1. Cell separation Human mononuclear leukocytes were obtained from heparinized blood from healthy young adult donors who were taking no medication at the time of 179
the study. Mononuclear leukocytes were separated by Ficoll/Uromiro (Pharmacia, Sweden and Bracco, Italy) sedimentation technique. Viability, measured by trypan blue exclusion, was more than 95%.
3.2. Cell cultures Mononuclear leukocytes were suspended in Tc-199 culture medium supplemented with 10% heat-inactivated calf serum, antibiotics and 25 mM HEPES (Serva, G.F.R.). Two-hundred/~1 of cell suspension containing 2 X 10 s viable cells were placed into flat-bottomed microplates (Greiner, G.F.R.), using 5 parallel samples. Con A was added to a final concentration of 25 #g/ml. The cultures were kept in humidified atmosphere for 72 h at 37°C. Eight hours before harvesting, 0.5/~Ci [3H] thymidine (Amersham, U.K.) was added to each well. Incorporation was terminated after 8 h and the cultures were sucked off onto glass-fiber filters (Whatman GF/C, U.K.). Isotope determinations were made in a Nuclear Chicago Isocap 300 counter. 3.3. Cell preincubation and treatment with PGE~ and indomethacin (1NDO) The freshly separated lymphocytes (106 cell/ml) were preincubated at 37°C for 24 h. The preincubated cells were cultured or treated with Con A, PGE1 and INDO according to the methods detailed above. PGEI (10 -6 M) and INDO (purchased from Sigma, U.S.A.; 1/ag/ml) were given at the start and at the 24th h of the lymphocyte culture. Con A was added to the cultures of freshly separated or preincubated lymphocytes at the start of culture (immediately after the cell separation, and after the 24 h preincubation). Duration of mitogen stimulation was always 72 h. The effect of PGE~ and INDO is expressed in percent of [3H] thymidine incorporation (cpm) of Con A-stimulated (control) cultures. Statistical significance was calculated by the Student's t-test. 3.4. Cyclic AMP determination The separated cells were suspended in Tc-199. The mononuclear leukocytes were treated with PGE1 (Sigma, U.S.A.) and with Con A (Calbiochem, U.S.A.) used at the optimal mitogenic concentration (25 ~g/ ml) for 10 min at 37 ° C. Cyclic AMP level was assessed by TRK-432 kit of Amersham (England) as detailed previously [15]. 180
4. Results 4.1. Effect o f preincubation on mitogen-stimulated DNA synthesis o f lymphocytes The DNA synthesis of Con A- (25/ag/ml) stimulated lymphocytes was enhanced when the reactive cells were precultured without mitogen for 24 h at 37°C (Fig. 1). After 24 h preincubation, the supernatants were removed, and the lymphocytes were resuspended and cultured in fresh culture nredium to avoid the effect of supernatant factors produced during preincubation. 4.2. Effect o f PGE 1 and INDO on the proliferation o f precultured lyrnphocytes PGE~ given simultaneously with Con A at the start of freshly separated lymphocyte culture inhibited the DNA synthesis. After 24 h culture of Con A-stimulated lymphocytes the PGE~ failed to inhibit the DNA synthesis. Preincubation of lymphocytes similarly abolished the effect of INDO (1/~g/ml) increasing DNA synthesis. When Con A and PGE~ or INDO were given simultaneously to the cells after 24 h preincubation, the PGE1 did not inhibit and INDO did not stinrulate the DNA synthesis of lymphocytes (Fig. 2). 4.3. PGE1 effect on intracellular cAMP level o f preincu bated mononuclear leu kocy tes After 24 h incubation at 37°C the intracellular %
p .<0.05
200 NS
T 100
Control
Con A
Fig. 1. Effect of 24 h preincubation at 37°C on the Con Astimulated DNA synthesis of human lymphocytes. Each column represents the mean of 8 experiments. 100¢~ = [3H ]thymidine incorporation of freshly separated lymphocytes. Control = thymidine incorporation without mitogen. ~, freshly separated cells; e, preincubated cells.
Oh 'i'
ConA PGEI'INDO
Oh 24 h
2/, h
Co:~ A
5. Discussion
Con A
PGEI,INDO
PGEI,INDO
/
.
.
.
.
.
.
:?
.
:,.oo
i
Fig. 2. Effect of PGE 1 (10 -6 M) and INDO (1 tsg/ml) on DNA synthesis of human lymphocytes. The substances were added immediately after the cell separation (0 h) or after 24 h preculture with or without Con A. 100% = cpm of [3H]thymidine incorporation of Con A-stimulated (control) culture; Con A = 25 ug/ml; n = 6; mean _+ S.E.M.
cAMP level markedly decreased. P G E 1 ( 1 0 -6 M)elevated the cAMP level of freshly separated cells. The preincubation at 37°C decreased the cAMP elevation exerted by PGE1. Con A alone did not influence the cAMP level. Con A slightly elevated the cAMP concentration in the PGEl-treated freshly separated cells (Fig. 3).
Oh pM
2/..h 37°C p(oool p
--~ p~oool
70
T,o~o1
50 \\\"
, NS <005 ,0O5
10, C o n t r Con A PGE
ConA+ PGEI
Contr. C o n A P G E I C o n A * PGE 1
Fig. 3. Effect of 24 h preincubation at 37°C on the cAMP level of PGE 1 and Con A-treated mononuclear leukocytes. PGE~ = 10 -6 M;Con A = 25 ug/ml; t = 10 m i n ; n = 6; cAMP pM/107 cells +_ S.E.M.
The prostaglandins elevating the intracellular cAMP level influence a range of immune reactions. PGE1 and -E2 inhibit the mitogen-induced lymphocyte proliferation in vitro. Lymphocyte subpopulations reveal different reactivity to PGE1 [23]. The human macrophages-monocytes [6,14,16,20], and according to animal experiments B [30], and T-lymphocytes [24] also produce prostaglandins. Prostaglandin production of cells can be inhibited by INDOblocking cyclo-oxygenase activity in the leukocytes. The prerequisite of PGE effect is that the immunocompetent cells, the lymphocytes [ 11 ] and the macrophages [ 13], have specific receptors in their plasma membranes. The distribution of PGE receptors among the human lymphocyte subpopulations is not homogenous [11] which might be an explanation for their functional heterogeneity. The PGE receptor is connected with the adenylcyclase in the plasma membrane. A suitable method to assess the PGE receptor function is to measure the PGE-induced elevation of intracellular cAMP level [1,3,4]. Bresnihan and Jasin [3] and Feighery et al. [6] gave a report on the "short-lived suppressor cell" function. According to their explanation, the suppressor cells lose their activity in the culture at 37°C resulting in the enhancement of lymphocyte proliferation after preincubation. We investigated the effect of 24 h preincubation at 37°C on Con A-induced DNA synthesis. The thymidine incorporation of preincubated lymphocytes was elevated significantly. This phenomenon cannot be explained by the function of "short-lived suppressor cells" (Kalm~ir et al., in preparation). This is in accordance with McCombs' data [19] who could not prove any suppressor activity of freshly separated lymphocytes on DNA synthesis of PHA or allogeneic cellstimulated lymphocytes. The enhancement of DNA synthesis after preincubation is a complex phenomenon which is in connection with the alteration of plasma membrane receptors on lymphocyte surfaces resulting in changes of intracellular cAMP level. PGE1 ( 1 0 -6 M ) i n h i b i t e d the mitogen stimulation by elevating the intracellular cAMP level. After preincubation, the PGE1 failed to decrease the DNA synthesis of lymphocytes. The effect of INDO increasing DNA synthesis of freshly separated lymphocytes 181
was similarly abolished after preincubation of cells. Both of these agents failed to influence DNA synthesis of lymphocytes precultured with or without Con A. These data are in accordance with those of Goodwin [11] and can be explained by the fact that the exogenous PGE~ and endogenous prostaglandins, the production of which can be inhibited by INDO, have no effect on preincubated lymphocytes because of the altered prostaglandin sensitivity of cells. It can be proved by our data that the PGE 1 causes smaller elevation of cAMP level in the preincubated cells, than in the fresh ones. The impairment of the PGE receptors is dependent on the temperature, and its function remains intact at 4°C (data not presented). PGE1 effect on intracellular cAMP level after preincubation at 37°C verify the data of PGE receptor "loss" found in the experiment showing the receptors by [3H] PGE~ [1 1]. Functional alteration of PGE receptors have recently been demonstrated on precultured animal peritoneal macrophages [ 13]. The mechanism of suppressive effect of PGE1 on DNA synthesis of lymphocytes has not been clarified, besides that its direct effect on proliferating ceils PGE~ may inhibit the production or effect on T-cell growth factor [ 1 ]. Decrease of PGE~ inhibitory effect having either direct or indirect influence upon cell proliferation can cause an elevation in DNA synthesis of preincubated lymphocytes. The cAMP level in the untreated cells was decreasing spontaneously during 24 h preincubation at 37°C. According to above data it is very likely that the change of effect of prostaglandins produced in the cultures and the spontaneous decrease of intracellular cAMP level explain the elevation of lymphocyte proliferation after preincubation. The cAMP increasing effect of PGE~ and some other substances stimulating adenylcyclase activity can be enhanced by mitogens [8,13]. We also measured a slight elevation of cAMP level when simultaneously with the PGE~ Con A was also added to the mononuclear leukocytes. The mechanism of this phenomenon can be explained by the cross-linkage of lymphocyte membrane structures caused by mitogen [31 ]. This effect may have a special significance because the (mitogen) stimulation sensitizes the lymphocytes toward the suppressor effect of prostaglandins, representing one of the models of feed-back suppressor regulation.
182
Acknowledgement We wish to thank Agnes Poly~k and Julia Czvikovszky for their expert technical assistance.
References [1 l Baker, P. E., Fahey, J. V. and Munck, A. (1981) Cell lmnmnol. 61,52 61. [2] Bourne, H. R., Lichtenstein, L. M., Mehnon, K. L., Itenney, C. S., Weinstein, Y. and Shearer, C. M. (1974) Science 184, 19-28. [3] Bresnihan, B. and Jasin, H. E. (1977) J. Clin. Invest. 59, 106 116. [4] Bromberg, S., Goodwin, J. S. and Peake, G. T. (1979) Clin. Res. 27, 36A. [51 Brunton, L. L., Wiklund, R. A., Van Arsdale, P. M. and Gihnan, A.G.(1976) J. Biol. Chem. 251,3037 3044. [61 Feighery, C., Whelan, C. A., Weir, D. G. and Greally, J.F.(1978) Clin. Exp. lmmunol. 32,459 465. [7] I:erraris, V. A. and DeRobertis, F. R. (1974) J. Clin. Invest. 54,378 386. [8] Gavin, J. R., Gorden, P., 111,Roth, J., Ash&, J. A. and Buell, O. (1973) J. Biol. Chem. 248, 2202-2207. [9] Gcmsa, D., Steggemann, L., Till, G. and Resch, K. (1977) J. Immunol. 119,524--529. [10] Goodwin, J. S., Bankhurst, A. D. and Messner, R. P. (1977) J. Exp. Med. 146, 1719 1734. [11] Goodwin, 1. S., Messner, R. P. and Peake, G. T. (1978) J. Clin. Invest. 62,753 760. [12] Goodwin, J. S., Viik, A., Lewis, M., Bankhurst, A. 1). and Williams, R. C. Jr. (1979) Cell hnmunol. 43, 150-159. [13] Goodwin, J. S., Kaszubowski, P. A. and Williams, R. C. Jr. (1979) J. Exp. Med. 150, 1260-1264. [ 14] Grunspan-Swirsky, A. and Pick, E. (1979) Cell Immunol. 45,415 427. [15] Humes, J. L., Bonney, R. J., Pelus, L. and Dahlgren, M.E.(1977) Nature (London) 269, 149 151. [ 16 ] Kalmdr, L., Ling, 1., Gergely, P., Fekete, B. and Petr~inyi. Gy. (1978) Acta Med. Acad. Sci. ttung. 35, 35 42. [171 Kurland, J. 1. and Bockman, R. (1978) J. Exp. Med. 147,952 957. [18] Lesniak, M.A.,Gorden, P.,Rotll, J. and Gavin, J.R.,Ill (1974) J. Biol. Chem. 249, 1661 1667. [19] McCombs, C. C., Michalski, J. P. and Talal, N. (1978) J. hnmunol. 120,532 537. [20] MacManus,J. P., Whitfield, J. F. and Youdala, T. (1971) J. Cell Physiol. 77, 103-116. [21] Passwell, J. H., Dayer, J. M. and Metier, E. (1979) 1. Immunol. 123,115 120. [ 22 ] Plescia, O. J., Yamamoto, I. and Shimanmra, T. (1975 ) Proc. Natl. Acad. Sci. U.S.A. 72,888 891.
[23] Rice, L., Laughter, A. and Twomey, J. J. (1979) J. Immunol. 1 2 2 , 9 9 1 - 9 9 6 . [24] Stobo, J. D., Kennedy, M. S. and Goldyne, M. E. (1979) J. Clin. Invest. 64, 1188-1195. [25] Strom, T. B., Lundin, A. P. and Carpenter, C. B. (1977) in: Progress in Clinical Immunology (Schwartz, R. S., ed.), Vol. 3, pp. 115 - 1 5 4 , Grune-Stratton, New York. [26] Webb, D. R. and Nowowiejski, 1. (1978) Cell Immunol. 41,72 85. [27] Webb, D. R., Stites, D. P., Perhnann, J. D., Austin, K. E. and Fudenberg, H. H. (1974) Clin. Immunol. Immunopath. 2 , 3 2 2 - 3 3 0 .
[28] Whitfield, J. F., MacManus, J. P. and Rixon, R. M. (1970a) J. Cell Physiol. 75,213 223. [29] Whitfield, J. F., MacManus, J. P. and Gillan, D. J. (1970b) J. Cell Physiol. 76, 65 70. [30] Whitfield, J. F., MacManus, J. P., Braceland, B. M. and Gillan, D. J. (1972) J. Cell Physiol. 79,353 362. [31] Williams, L. T., Snyderman, R. and Lefkowitz, R. J. (1976) J. Clin. Invest. 5 7 , 1 4 9 - 1 5 5 . [32] Zimecki, M. and Webb, D. R. (1976) J. Immunol. 117, 2158 2164. [33] Yahara, 1. and Edelman, G. M. (1973) Exp. Cell Res. 81,143-155.
183
Elsevier BiomedicalPress
ENHANCEMENT OF MITOGEN-INDUCED LYMPHOCYTE PROLIFERATION AFTER P R E I N C U B A T I O N IS DUE T O A L T E R E D S E N S I T I V I T Y T O P R O S T A G L A N D I N S L. KALMAR and P. GERGELY 2nd Department o f Medicine, Semmelweis University, Budapest, Hungary
(Received 1 September 1981) (Accepted 5 December 1981)
1. Summary Twenty-four hour preincubation at 37°C enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolishes the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37°C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37°C, PGE~ elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGEI and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37°C.
There is a large body of evidence on the immunological effects of prostaglandins. The prostaglandins may have a direct role in the regulation of immunological processes [9,21,22]. A connection has been assumed between the prostaglandin-producing suppressor cell function and the so-called "short-lived suppressor cell" phenomenon [10]. Prerequisites of the regulatory effects of prostaglandins are the following: (I) immunocompetent cells are able to produce prostaglandins (mostly PGE1 and PGE2), and the rate of production can be influenced by antigens and mitogens [6,16,19,25 ]; and (2) the effector cell functions can be altered by the produced prostaglandins because these cells have specific prostaglandin receptors in their plasma membranes. The PGE~ receptor function of human peripheral blood mononuclear leukocytes was investigated after 24 h preincubation at 37°C. The preincubation technique was identical to that used in the "short-lived suppressor cell" test [2,5]. Our aim was to study the effect of preincubation on the "spontaneous" intracellular cyclic adenosine-3',5'-monophosphate (cAMP) level and cAMP level of cells treated with PGE1 and the connection of the above parameters with the change of mitogen-induced lymphocyte proliferation after preincubation.
3. Materials and methods
2. Introduction kymphocytes have receptors for many hormones and hormone4ike substances [7,17,18,26,27,29]. Their function and biological role are not well known. 0165-2478/82/0000-0000/$2.75 © ElsevierBiomedicalPress
3.1. Cell separation Human mononuclear leukocytes were obtained from heparinized blood from healthy young adult donors who were taking no medication at the time of 179
the study. Mononuclear leukocytes were separated by Ficoll/Uromiro (Pharmacia, Sweden and Bracco, Italy) sedimentation technique. Viability, measured by trypan blue exclusion, was more than 95%.
3.2. Cell cultures Mononuclear leukocytes were suspended in Tc-199 culture medium supplemented with 10% heat-inactivated calf serum, antibiotics and 25 mM HEPES (Serva, G.F.R.). Two-hundred/~1 of cell suspension containing 2 X 10 s viable cells were placed into flat-bottomed microplates (Greiner, G.F.R.), using 5 parallel samples. Con A was added to a final concentration of 25 #g/ml. The cultures were kept in humidified atmosphere for 72 h at 37°C. Eight hours before harvesting, 0.5/~Ci [3H] thymidine (Amersham, U.K.) was added to each well. Incorporation was terminated after 8 h and the cultures were sucked off onto glass-fiber filters (Whatman GF/C, U.K.). Isotope determinations were made in a Nuclear Chicago Isocap 300 counter. 3.3. Cell preincubation and treatment with PGE~ and indomethacin (1NDO) The freshly separated lymphocytes (106 cell/ml) were preincubated at 37°C for 24 h. The preincubated cells were cultured or treated with Con A, PGE1 and INDO according to the methods detailed above. PGEI (10 -6 M) and INDO (purchased from Sigma, U.S.A.; 1/ag/ml) were given at the start and at the 24th h of the lymphocyte culture. Con A was added to the cultures of freshly separated or preincubated lymphocytes at the start of culture (immediately after the cell separation, and after the 24 h preincubation). Duration of mitogen stimulation was always 72 h. The effect of PGE~ and INDO is expressed in percent of [3H] thymidine incorporation (cpm) of Con A-stimulated (control) cultures. Statistical significance was calculated by the Student's t-test. 3.4. Cyclic AMP determination The separated cells were suspended in Tc-199. The mononuclear leukocytes were treated with PGE1 (Sigma, U.S.A.) and with Con A (Calbiochem, U.S.A.) used at the optimal mitogenic concentration (25 ~g/ ml) for 10 min at 37 ° C. Cyclic AMP level was assessed by TRK-432 kit of Amersham (England) as detailed previously [15]. 180
4. Results 4.1. Effect o f preincubation on mitogen-stimulated DNA synthesis o f lymphocytes The DNA synthesis of Con A- (25/ag/ml) stimulated lymphocytes was enhanced when the reactive cells were precultured without mitogen for 24 h at 37°C (Fig. 1). After 24 h preincubation, the supernatants were removed, and the lymphocytes were resuspended and cultured in fresh culture nredium to avoid the effect of supernatant factors produced during preincubation. 4.2. Effect o f PGE 1 and INDO on the proliferation o f precultured lyrnphocytes PGE~ given simultaneously with Con A at the start of freshly separated lymphocyte culture inhibited the DNA synthesis. After 24 h culture of Con A-stimulated lymphocytes the PGE~ failed to inhibit the DNA synthesis. Preincubation of lymphocytes similarly abolished the effect of INDO (1/~g/ml) increasing DNA synthesis. When Con A and PGE~ or INDO were given simultaneously to the cells after 24 h preincubation, the PGE1 did not inhibit and INDO did not stinrulate the DNA synthesis of lymphocytes (Fig. 2). 4.3. PGE1 effect on intracellular cAMP level o f preincu bated mononuclear leu kocy tes After 24 h incubation at 37°C the intracellular %
p .<0.05
200 NS
T 100
Control
Con A
Fig. 1. Effect of 24 h preincubation at 37°C on the Con Astimulated DNA synthesis of human lymphocytes. Each column represents the mean of 8 experiments. 100¢~ = [3H ]thymidine incorporation of freshly separated lymphocytes. Control = thymidine incorporation without mitogen. ~, freshly separated cells; e, preincubated cells.
Oh 'i'
ConA PGEI'INDO
Oh 24 h
2/, h
Co:~ A
5. Discussion
Con A
PGEI,INDO
PGEI,INDO
/
.
.
.
.
.
.
:?
.
:,.oo
i
Fig. 2. Effect of PGE 1 (10 -6 M) and INDO (1 tsg/ml) on DNA synthesis of human lymphocytes. The substances were added immediately after the cell separation (0 h) or after 24 h preculture with or without Con A. 100% = cpm of [3H]thymidine incorporation of Con A-stimulated (control) culture; Con A = 25 ug/ml; n = 6; mean _+ S.E.M.
cAMP level markedly decreased. P G E 1 ( 1 0 -6 M)elevated the cAMP level of freshly separated cells. The preincubation at 37°C decreased the cAMP elevation exerted by PGE1. Con A alone did not influence the cAMP level. Con A slightly elevated the cAMP concentration in the PGEl-treated freshly separated cells (Fig. 3).
Oh pM
2/..h 37°C p(oool p
--~ p~oool
70
T,o~o1
50 \\\"
, NS <005 ,0O5
10, C o n t r Con A PGE
ConA+ PGEI
Contr. C o n A P G E I C o n A * PGE 1
Fig. 3. Effect of 24 h preincubation at 37°C on the cAMP level of PGE 1 and Con A-treated mononuclear leukocytes. PGE~ = 10 -6 M;Con A = 25 ug/ml; t = 10 m i n ; n = 6; cAMP pM/107 cells +_ S.E.M.
The prostaglandins elevating the intracellular cAMP level influence a range of immune reactions. PGE1 and -E2 inhibit the mitogen-induced lymphocyte proliferation in vitro. Lymphocyte subpopulations reveal different reactivity to PGE1 [23]. The human macrophages-monocytes [6,14,16,20], and according to animal experiments B [30], and T-lymphocytes [24] also produce prostaglandins. Prostaglandin production of cells can be inhibited by INDOblocking cyclo-oxygenase activity in the leukocytes. The prerequisite of PGE effect is that the immunocompetent cells, the lymphocytes [ 11 ] and the macrophages [ 13], have specific receptors in their plasma membranes. The distribution of PGE receptors among the human lymphocyte subpopulations is not homogenous [11] which might be an explanation for their functional heterogeneity. The PGE receptor is connected with the adenylcyclase in the plasma membrane. A suitable method to assess the PGE receptor function is to measure the PGE-induced elevation of intracellular cAMP level [1,3,4]. Bresnihan and Jasin [3] and Feighery et al. [6] gave a report on the "short-lived suppressor cell" function. According to their explanation, the suppressor cells lose their activity in the culture at 37°C resulting in the enhancement of lymphocyte proliferation after preincubation. We investigated the effect of 24 h preincubation at 37°C on Con A-induced DNA synthesis. The thymidine incorporation of preincubated lymphocytes was elevated significantly. This phenomenon cannot be explained by the function of "short-lived suppressor cells" (Kalm~ir et al., in preparation). This is in accordance with McCombs' data [19] who could not prove any suppressor activity of freshly separated lymphocytes on DNA synthesis of PHA or allogeneic cellstimulated lymphocytes. The enhancement of DNA synthesis after preincubation is a complex phenomenon which is in connection with the alteration of plasma membrane receptors on lymphocyte surfaces resulting in changes of intracellular cAMP level. PGE1 ( 1 0 -6 M ) i n h i b i t e d the mitogen stimulation by elevating the intracellular cAMP level. After preincubation, the PGE1 failed to decrease the DNA synthesis of lymphocytes. The effect of INDO increasing DNA synthesis of freshly separated lymphocytes 181
was similarly abolished after preincubation of cells. Both of these agents failed to influence DNA synthesis of lymphocytes precultured with or without Con A. These data are in accordance with those of Goodwin [11] and can be explained by the fact that the exogenous PGE~ and endogenous prostaglandins, the production of which can be inhibited by INDO, have no effect on preincubated lymphocytes because of the altered prostaglandin sensitivity of cells. It can be proved by our data that the PGE 1 causes smaller elevation of cAMP level in the preincubated cells, than in the fresh ones. The impairment of the PGE receptors is dependent on the temperature, and its function remains intact at 4°C (data not presented). PGE1 effect on intracellular cAMP level after preincubation at 37°C verify the data of PGE receptor "loss" found in the experiment showing the receptors by [3H] PGE~ [1 1]. Functional alteration of PGE receptors have recently been demonstrated on precultured animal peritoneal macrophages [ 13]. The mechanism of suppressive effect of PGE1 on DNA synthesis of lymphocytes has not been clarified, besides that its direct effect on proliferating ceils PGE~ may inhibit the production or effect on T-cell growth factor [ 1 ]. Decrease of PGE~ inhibitory effect having either direct or indirect influence upon cell proliferation can cause an elevation in DNA synthesis of preincubated lymphocytes. The cAMP level in the untreated cells was decreasing spontaneously during 24 h preincubation at 37°C. According to above data it is very likely that the change of effect of prostaglandins produced in the cultures and the spontaneous decrease of intracellular cAMP level explain the elevation of lymphocyte proliferation after preincubation. The cAMP increasing effect of PGE~ and some other substances stimulating adenylcyclase activity can be enhanced by mitogens [8,13]. We also measured a slight elevation of cAMP level when simultaneously with the PGE~ Con A was also added to the mononuclear leukocytes. The mechanism of this phenomenon can be explained by the cross-linkage of lymphocyte membrane structures caused by mitogen [31 ]. This effect may have a special significance because the (mitogen) stimulation sensitizes the lymphocytes toward the suppressor effect of prostaglandins, representing one of the models of feed-back suppressor regulation.
182
Acknowledgement We wish to thank Agnes Poly~k and Julia Czvikovszky for their expert technical assistance.
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